Molecular Biology Studies On The Different Phenotypes Of Fruiting-body Forming Of Cordyceps Militaris

Cordyceps militaris(L.) Link is a species of Cordyceps which can parasit in the pupas and lavae of many species of Lepidoptera. It exerts fine value in the fields of functional foods, medicine and biological technology. Recent years some functional foods & medicine which named Cordyceps sinensis are produced by Cordyceps militaris. Artificial culture Cordyceps militaris is the well way to solve the insufficient resource of wild Cordyceps militaris. In the process of Artificial culture Cordyceps militaris, the degeneration phenomenon of the strains is the restrictive bottleneck of the industrial exploitation. At present,there is few reports to aim directly at systematic molecular biology research of heterokaryon and mating type of Cordyceps militaris, but these two aspects can well reveal the essence of degeneration specifically.In the beginning of this experiment, a superior initial strain of Cordyceps militaris (Number:WWM04)was isolated single spore. The 22 single spore isolation strains were obtained. It was found that 20 of them were no fruiting-body forming strains and 2 of them were fruiting-body forming strains after cultivated the 22 single spore isolation strains on the cooked ice medium. To observe colony feature cultivated on PDA medium & mycelial feature by shake cultivation of the 6 no fruiting-body forming single spore isolation strains, the degenerated strains from 12 tansfer arid initial strains, it indicated that there existed differences not only between them and the initial superior strains& the degenerated strains from 12 tansfe, but also among these 6 no fruiting-body forming single spore isolation colonial morphology themselves. It is the typical pleomorphism of Cordyceps militaris.The second part of the experiment, used the RAPD technique and rDNA ITS sequence analysis to analyse the 4 the no fruiting-body forming single spore isolation strains, the 1 fruiting-body forming single spore isolation strains,the degenerated strains from 12 tansfer and initial strains of Cordyceps militaris. The UPGMA dendrogram constructed by RAPD analysis and branch dendrogram of molecular system based on ITS sequence. It indicated that there existed visible differences among the RAPDs atlas of these no fruiting-body forming single spore isolation strains,the initial superior strains and the transferred degenerated strains, but the difference of the initial superior strains and the transferred degenerated strains is less; the difference of the ITS sequences is little among all the strains. All the result indicate the main reason of the serious phenomenon of degeneration of Cordyceps militaris is difference of chromosome by parasexual reproduction.The third part of the experiment, to design primers of two most conserved mating-type genes( MAT1-1-1,MAT1-2-1) , and grope the optimization reaction codition to carry out the study of mating-type of the 16 no fruiting-body forming single spore isolation strains, the 2 fruiting-body forming single spore isolation strains, 1 degenerative strain from 12 transfers, and the initial strain. Results indicate that all no fruiting-body forming single spore isolation strains have single mating -type, either MAT1-1 or MAT1-2. But all fruit body single spore isolation strains have both MAT1-1 and MAT1-2. It illustrated that Cordyceps militaris is a heterothallic fungus. Based on MAT1-2-1 mating-type gene sequence phylogenetic analyzing, it is found that there existed accordance between the phylogenetic trees constructed by MAT1-2-1 mating-type gene sequence and the ITS sequence, the two types of phylogenetic trees can both used in phylogenetic analyzing.